De Novo Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials.

Metagenomic sequences represent an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequences requires the identification of suitable candidate systems as starting scaffolds for de novo DNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic "trash bin" for genes associated with conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines. Using a human metagenomic gut data set representing 2805 taxonomically distinct units, we identified 1598 contigs containing conjugation genes with a differential distribution in human cohorts. We synthesized de novo an entire Citrobacter spp. conjugative system of 54 kb containing at least 47 genes and assembled it into a plasmid, pCitro. We found that pCitro conjugates from Escherichia coli to Citrobacter rodentium with a 30-fold higher frequency than to E. coli, and is compatible with Citrobacter resident plasmids. Mutations in the traV and traY conjugation components of pCitro inhibited conjugation. We showed that pCitro can be repurposed as an antimicrobial delivery agent by programming it with the TevCas9 nuclease and Citrobacter-specific sgRNAs to kill C. rodentium. Our study reveals a trove of uncharacterized conjugative systems in metagenomic data and describes an experimental framework to animate these large genetic systems as novel target-adapted delivery vectors for Cas9-based editing of bacterial genomes.

Blockade or deficiency of PD-L1 expression in intestinal allograft accelerates graft tissue injury in mice.

The importance of PD-1/PD-L1 interaction to alloimmune response is unknown in intestinal transplantation. We tested whether PD-L1 regulates allograft tissue injury in murine intestinal transplantation. PD-L1 expression was observed on the endothelium and immune cells in the intestinal allograft. Monoclonal antibody treatment against PD-L1 led to accelerated allograft tissue damage, characterized by severe cellular infiltrations, massive destruction of villi, and increased crypt apoptosis in the graft. Interestingly, PD-L1-/- allografts were more severely rejected than wild-type allografts, but the presence or absence of PD-L1 in recipients did not affect the degree of allograft injury. PD-L1-/- allografts showed increased infiltrating Ly6G+ and CD11b+ cells in lamina propria on day 4, whereas the degree of CD4+ or CD8+ T cell infiltration was comparable to wild-type allografts. Gene expression analysis revealed that PD-L1-/- allografts had increased mRNA expressions of Cxcr2, S100a8/9, Nox1, IL1rL1, IL1r2, and Nos2 in the lamina propria cells on day 4. Taken together, study results suggest that PD-L1 expression in the intestinal allograft, but not in the recipient, plays a critical role in mitigating allograft tissue damage in the early phase after transplantation. The PD-1/PD-L1 interaction may contribute to immune regulation of the intestinal allograft via the innate immune system.

Pharmacological inhibition of BACE1 suppresses glioblastoma growth by stimulating macrophage phagocytosis of tumor cells.

Glioblastoma (GBM) contains abundant tumor-associated macrophages (TAMs). The majority of TAMs are tumor-promoting macrophages (pTAMs), while tumor-suppressive macrophages (sTAMs) are the minority. Thus, reprogramming pTAMs into sTAMs represents an attractive therapeutic strategy. By screening a collection of small-molecule compounds, we find that inhibiting ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) with MK-8931 potently reprograms pTAMs into sTAMs and promotes macrophage phagocytosis of glioma cells; moreover, low-dose radiation markedly enhances TAM infiltration and synergizes with MK-8931 treatment to suppress malignant growth. BACE1 is preferentially expressed by pTAMs in human GBMs and is required to maintain pTAM polarization through trans-interleukin 6 (IL-6)-soluble IL-6 receptor (sIL-6R)-signal transducer and activator of transcription 3 (STAT3) signaling. Because MK-8931 and other BACE1 inhibitors have been developed for Alzheimer's disease and have been shown to be safe for humans in clinical trials, these inhibitors could potentially be streamlined for cancer therapy. Collectively, this study offers a promising therapeutic approach to enhance macrophage-based therapy for malignant tumors.

Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing.

The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. CRISPR nucleases can be programmed to kill bacteria, but require an efficient and broad-host range delivery system to be effective. Here, using an Escherichia coli and Salmonella enterica co-culture system, we show that plasmids based on the IncP RK2 conjugative system can be used as delivery vectors for a TevSpCas9 dual nuclease. Notably, a cis-acting plasmid that encodes the conjugation and CRISPR machinery conjugates from E. coli to S. enterica with high frequency compared to a trans system that separates conjugation and CRISPR machinery. In culture conditions that enhance cell-to-cell contact, conjugation rates approach 100% with the cis-acting plasmid. Targeting of single or multiplexed sgRNAs to non-essential genes results in high S. enterica killing efficiencies. Our data highlight the potential of cis-acting conjugative plasmids as a delivery system for CRISPR nucleases or other microbial-altering agents for targeted bacterial killing.

IL-17R-EGFR axis links wound healing to tumorigenesis in Lrig1+ stem cells.

Lrig1 marks a distinct population of stem cells restricted to the upper pilosebaceous unit in normal epidermis. Here we report that IL-17A-mediated activation of EGFR plays a critical role in the expansion and migration of Lrig1+ stem cells and their progenies in response to wounding, thereby promoting wound healing and skin tumorigenesis. Lrig1-specific deletion of the IL-17R adaptor Act1 or EGFR in mice impairs wound healing and reduces tumor formation. Mechanistically, IL-17R recruits EGFR for IL-17A-mediated signaling in Lrig1+ stem cells. While TRAF4, enriched in Lrig1+ stem cells, tethers IL-17RA and EGFR, Act1 recruits c-Src for IL-17A-induced EGFR transactivation and downstream activation of ERK5, which promotes the expansion and migration of Lrig1+ stem cells. This study demonstrates that IL-17A activates the IL-17R-EGFR axis in Lrig1+ stem cells linking wound healing to tumorigenesis.

Neuron-Specific HuR-Deficient Mice Spontaneously Develop Motor Neuron Disease.

Human Ag R (HuR) is an RNA binding protein in the ELAVL protein family. To study the neuron-specific function of HuR, we generated inducible, neuron-specific HuR-deficient mice of both sexes. After tamoxifen-induced deletion of HuR, these mice developed a phenotype consisting of poor balance, decreased movement, and decreased strength. They performed significantly worse on the rotarod test compared with littermate control mice, indicating coordination deficiency. Using the grip-strength test, it was also determined that the forelimbs of neuron-specific HuR-deficient mice were much weaker than littermate control mice. Immunostaining of the brain and cervical spinal cord showed that HuR-deficient neurons had increased levels of cleaved caspase-3, a hallmark of cell apoptosis. Caspase-3 cleavage was especially strong in pyramidal neurons and α motor neurons of HuR-deficient mice. Genome-wide microarray and real-time PCR analysis further indicated that HuR deficiency in neurons resulted in altered expression of genes in the brain involved in cell growth, including trichoplein keratin filament-binding protein, Cdkn2c, G-protein signaling modulator 2, immediate early response 2, superoxide dismutase 1, and Bcl2. The additional enriched Gene Ontology terms in the brain tissues of neuron-specific HuR-deficient mice were largely related to inflammation, including IFN-induced genes and complement components. Importantly, some of these HuR-regulated genes were also significantly altered in the brain and spinal cord of patients with amyotrophic lateral sclerosis. Additionally, neuronal HuR deficiency resulted in the redistribution of TDP43 to cytosolic granules, which has been linked to motor neuron disease. Taken together, we propose that this neuron-specific HuR-deficient mouse strain can potentially be used as a motor neuron disease model.

IL-17-receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling.

Mechanisms that degrade inflammatory mRNAs are well known; however, stabilizing mechanisms are poorly understood. Here, we show that Act1, an interleukin-17 (IL-17)-receptor-complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, the SEFIR-binding element (SBE), in the 3' untranslated region (UTR) of Cxcl1 mRNA, encoding an inflammatory chemokine. mRNA-bound Act1 directs formation of three compartmentally distinct RNA-protein complexes (RNPs) that regulate three disparate events in inflammatory-mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P bodies and promoting translation. SBE RNA aptamers decreased IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, thus providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3' UTRs of select mRNAs and consequently controls receptor-mediated mRNA stabilization and translation during inflammation.

IL-17A-Induced PLET1 Expression Contributes to Tissue Repair and Colon Tumorigenesis.

This study identifies a novel mechanism linking IL-17A with colon tissue repair and tumor development. Abrogation of IL-17A signaling in mice attenuated tissue repair of dextran sulfate sodium (DSS)-induced damage in colon epithelium and markedly reduced tumor development in an azoxymethane/DSS model of colitis-associated cancer. A novel IL-17A target gene, PLET1 (a progenitor cell marker involved in wound healing), was highly induced in DSS-treated colon tissues and tumors in an IL-17RC-dependent manner. PLET1 expression was induced in LGR5+ colon epithelial cells after DSS treatment. LGR5+PLET1+ marks a highly proliferative cell population with enhanced expression of IL-17A target genes. PLET1 deficiency impaired tissue repair of DSS-induced damage in colon epithelium and reduced tumor formation in an azoxymethane/DSS model of colitis-associated cancer. Our results suggest that IL-17A-induced PLET1 expression contributes to tissue repair and colon tumorigenesis.

IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.

Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

TRPV4 Mechanosensitive Ion Channel Regulates Lipopolysaccharide-Stimulated Macrophage Phagocytosis.

Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens, and the extracellular matrix. In the present study we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the LPS and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of nonopsonized particles (Escherichia coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/proresolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.

A novel IL-17 signaling pathway controlling keratinocyte proliferation and tumorigenesis via the TRAF4-ERK5 axis.

Although IL-17 is emerging as an important cytokine in cancer promotion and progression, the underlining molecular mechanism remains unclear. Previous studies suggest that IL-17 (IL-17A) sustains a chronic inflammatory microenvironment that favors tumor formation. Here we report a novel IL-17-mediated cascade via the IL-17R-Act1-TRAF4-MEKK3-ERK5 positive circuit that directly stimulates keratinocyte proliferation and tumor formation. Although this axis dictates the expression of target genes Steap4 (a metalloreductase for cell metabolism and proliferation) and p63 (a transcription factor for epidermal stem cell proliferation), Steap4 is required for the IL-17-induced sustained expansion of p63(+) basal cells in the epidermis. P63 (a positive transcription factor for the Traf4 promoter) induces TRAF4 expression in keratinocytes. Thus, IL-17-induced Steap4-p63 expression forms a positive feedback loop through p63-mediated TRAF4 expression, driving IL-17-dependent sustained activation of the TRAF4-ERK5 axis for keratinocyte proliferation and tumor formation.

Myeloid colony-stimulating factors as regulators of macrophage polarization.

The scope of functional heterogeneity in macrophages has been defined by two polarized end states known as M1 and M2, which exhibit the proinflammatory activities necessary for host defense and the tissue repair activities required for restoration of homeostasis, respectively. Macrophage populations in different tissue locations exist in distinct phenotypic states across this M1/M2 spectrum and the development and abundance of individual subsets result from the local and systemic action of myeloid colony-stimulating factors (CSFs) including M-CSF and GM-CSF. These factors have relatively non-overlapping roles in the differentiation and maintenance of specific macrophage subsets. Furthermore, there is now evidence that CSFs may also regulate macrophage phenotype during challenge. Cell culture studies from multiple laboratories demonstrate that macrophages developed in the presence of GM-CSF exhibit amplified response to M1 polarizing stimuli while M-CSF potentiates responses to M2 stimuli. As a consequence, these factors can be important determinants of the magnitude and duration of both acute and chronic inflammatory pathology and may, therefore, be potential targets for therapeutic manipulation in specific human disease settings.

Cellular stress amplifies TLR3/4-induced CXCL1/2 gene transcription in mononuclear phagocytes via RIPK1.

The impact of environmental stressors on the magnitude of specific chemokine gene expression was examined in mouse bone marrow-derived macrophages stimulated through various TLRs. Levels of TLR-stimulated CXCL1 and CXCL2 but not CXCL10 or CCL5 mRNAs were selectively enhanced (>10-fold) in stressed macrophages. The amplification was also manifested for other proinflammatory cytokines, including TNF-α, IL-1α, and IL-6. Responses through TLR3 and TLR4 exhibited the greatest sensitivity, reflecting a requirement for Toll/IL-IR domain-containing adaptor-inducing IFN-ß (TRIF), the adaptor protein selectively associated with these TLRs. IFN regulatory factor 3, a transcription factor that is downstream of TLR4/TRIF signaling, was not required for sensitivity to stress-induced chemokine amplification. c/EBP homologous protein and X box binding protein 1 have been reported to enhance inflammatory cytokine responses but are not required for amplification of TLR3/4-induced CXCL1 expression. Rather, receptor-interacting protein kinase 1, a kinase also linked with TLR3/4/TRIF signaling, is required and involves a stress-dependent increase in its abundance and ubiquitination. Whereas NF-κB activation is necessary for TLR-induced chemokine gene transcription, this factor does not appear to be the primary mechanistic target of environmental stress. The application of stress also enhanced chemokine expression in macrophages infiltrating the peritoneal cavity but was not observed in the resident peritoneal cells or in the liver. These findings identify novel mechanisms for modulating the magnitude and duration of selective TLR-induced chemokine and cytokine gene expression and further establish the importance of cell stress pathways in coordinating the outcomes of cellular and tissue injury.

All-trans retinoic acid induces arginase-1 and inducible nitric oxide synthase-producing dendritic cells with T cell inhibitory function.

Hepatic stellate cells (HSC) are a major source of the immunoregulatory metabolite all-trans retinoic acid (ATRA), which may contribute to the generation of tolerogenic dendritic cells (DCs) in the liver. The present study seeks to clarify the mechanism(s) through which ATRA promotes the development of tolerogenic DCs. Although bone marrow-derived ATRA-treated DCs (RA-DCs) and conventional DCs had comparable surface phenotype, RA-DCs had diminished stimulatory capacity and could directly inhibit the expansion of DC/OVA-stimulated OT-II T cells. Arginase-1 (Arg-1) was found promote suppression because 1) ATRA was a potent inducer of Arg-1 protein and activity, 2) the Arg-1 inhibitor N(w)-hydroxy nor-l-arginine partially reversed suppression, and 3) the suppressive function of RA-DCs was partially compromised using OT-II T cells from GCN2(-/-) mice, which are insensitive to Arg-1. Inducible NO synthase (iNOS), however, was found to be a more significant contributor to RA-DC function because 1) ATRA potentiated the expression of IFN-γ-induced iNOS, 2) suppressive function in RA-DCs was blocked by the iNOS inhibitor N(G)-monomethyl-l-arginine, monoacetate salt, and 3) RA-DCs derived from iNOS(-/-) mice exhibited near complete loss of tolerogenic function, despite sustained Arg-1 activity. The expression of iNOS and the suppressive function of RA-DCs were dependent on both IFN-γ and ATRA. Furthermore, the in vivo behavior of RA-DCs proved to be consistent with their in vitro behavior. Thus, we conclude that ATRA enhances both Arg-1 and iNOS expression in IFN-γ-treated DCs, resulting in a tolerogenic phenotype. These findings elucidate mechanisms through which ATRA may contribute to liver immune tolerance.

HuR is required for IL-17-induced Act1-mediated CXCL1 and CXCL5 mRNA stabilization.

IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA-binding protein HuR in IL-17-induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17-induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. Although IL-17 induced the coshift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translation-active/translation-inactive IL-17-induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17-induced neutrophilia. In summary, HuR functions to couple receptor-proximal signaling to posttranscriptional machinery, contributing to IL-17-induced inflammation.

Diversity in sequence-dependent control of GRO chemokine mRNA half-life.

Neutrophil trafficking to sites of injury or infection is regulated, in part, by the closely related GRO family of chemokines (CXCL1, -2, and -3). Expression of the GRO chemokine genes is known to be determined by transcriptional bursts in response to proinflammatory stimulation, but post-transcriptional mechanisms that regulate mRNA half-life are now recognized as important determinants. mRNA half-life is regulated via distinct sequence motifs and sequence-specific, RNA-binding proteins, whose function is subject to regulation by extracellular proinflammatory stimuli. Moreover, such mechanisms exhibit cell-type and stimulus dependency. We now present evidence that in nonmyeloid cells, GRO2 and GRO3 isoforms exhibit at least two patterns of mRNA instability that are distinguished by differential sensitivity to specific mRNA-destabilizing proteins and stimulus-mediated prolongation of mRNA half-life, respectively. Although the 3' UTR regions of GRO2 and GRO3 mRNAs contain multiple AREs, GRO2 has eight AUUUA pentamers, whereas GRO3 has seven. These confer quantitative differences in half-life and show sensitivity for TTP and KSRP but not SF2/ASF. Moreover, these AUUUA determinants do not confer instability that can be modulated in response to IL-1α. In contrast, IL-1α-sensitive instability for GRO2 and GRO3 is conferred by sequences located proximal to the 3' end of the 3'UTR that are independent of the AUUUA sequence motif. These regions are insensitive to TTP and KSRP but show reduced half-life mediated by SF2/ASF. These sequence-linked, post-transcriptional activities provide substantial mechanistic diversity in the control of GRO family chemokine gene expression.

A CC' loop decoy peptide blocks the interaction between Act1 and IL-17RA to attenuate IL-17- and IL-25-induced inflammation.

Interleukin-17 (IL-17) and IL-25 signaling induce the expression of genes encoding inflammatory factors and are implicated in the pathology of various inflammatory diseases. Nuclear factor κB (NF-κB) activator 1 (Act1) is an adaptor protein and E3 ubiquitin ligase that is critical for signaling by either IL-17 or IL-25, and it is recruited to their receptors (IL-17R and IL-25R) through heterotypic interactions between the SEFIR [SEF (similar expression to fibroblast growth factor genes) and IL-17R] domain of Act1 and that of the receptor. SEFIR domains have structural similarity with the Toll-IL-1 receptor (TIR) domains of Toll-like receptors and IL-1R. Whereas the BB' loop of TIR is required for TIR-TIR interactions, we found that deletion of the BB' loop from Act1 or IL-17RA (a common subunit of both IL-17R and IL-25R) did not affect Act1-IL-17RA interactions; rather, deletion of the CC' loop from Act1 or IL-17RA abolished the interaction between both proteins. Surface plasmon resonance measurements showed that a peptide corresponding to the CC' loop of Act1 bound directly to IL-17RA. A cell-permeable decoy peptide based on the CC' loop sequence inhibited IL-17- or IL-25-mediated signaling in vitro, as well as IL-17- and IL-25-induced pulmonary inflammation in mice. Together, these findings provide the molecular basis for the specificity of SEFIR-SEFIR versus TIR-TIR domain interactions and consequent signaling. Moreover, we suggest that the CC' loop motif of SEFIR domains is a promising target for therapeutic strategies against inflammatory diseases associated with IL-17 or IL-25 signaling.

IL-17 signaling for mRNA stabilization does not require TNF receptor-associated factor 6.

IL-17 alone is a relatively weak inducer of gene expression, but cooperates with other cytokines, including TNF-alpha, to generate a strong response in part via prolongation of mRNA t(1/2). Because TNFR-associated factor 6 (TRAF6) has been reported to be essential for signaling by IL-17, we examined its involvement in IL-17-mediated mRNA stabilization. Although overexpression of TRAF6 in HeLa cells activates NF-kappaB, it does not stabilize transfected KC mRNA. Furthermore, a dominant-negative TRAF6 abrogates NF-kappaB activation, but does not block IL-17-induced chemokine mRNA stabilization. IL-17 can stabilize KC and MIP-2 mRNAs comparably in TNF-alpha-treated mouse embryo fibroblasts from TRAF6(+/+) and TRAF6(-/-) mice. TRAF6 is known to couple upstream signals with activation of p38 MAPK and mitogen activated protein kinase activated protein kinase 2, both of which have been shown to be important for Toll/IL-1R-mediated mRNA stabilization in various cell types. Inhibition of p38 MAPK, however, does not block IL-17-induced KC mRNA stabilization, and IL-17 can stabilize KC mRNA equally in mouse embryo fibroblasts from both wild-type and mitogen activated protein kinase activated protein kinase 2/3 doubly-deficient mice. Finally, IL-17 can amplify the levels of multiple TNF-alpha-stimulated mRNAs in wild-type and TRAF6-deficient cells, but not in cells from Act1(-/-) mice. Collectively, these findings demonstrate the existence of a TRAF6/p38 MAPK-independent pathway that couples the IL-17R with enhanced mRNA stability. Because the most potent effects of IL-17 on gene expression are obtained in cooperation with other cytokines such as TNF-alpha, these findings suggest that this pathway is a major contributing mechanism for response to IL-17.